Explanation 1 . Rates of change in FEV1 and DLCO as potential indicators for mTOR inhibitor therapy in premenopausal lymphangioleiomyomatosis (LAM) patients The MILES trial showed that sirolimus stabilises lung function in patients with forced expiratory volume in 1 s (FEV1) 70% predicted. Isolated reduction in diffusing capacity of the lung for carbon monoxide (DLCO) 70% was not a criterion for enrolment. Accordingly, the MILES trial criteria for initiation of mTOR inhibitor therapy excluded patients with FEV1 >70%, who, by the time their FEV1 declined to 70% pred, might have lost one-third or more of their lung function. Although there is general agreement that LAM patients with chylous effusions and lymphangioleiomyomas and angiomyolipomas benefit from mTOR inhibitor therapy, no criteria based on FEV1 or DLCO and their rates of decline have been proposed to identify LAM patients at risk for disease progression who may benefit from earlier initiation of mTOR inhibitor therapy, except those derived from the MILES trial. The problem with identifying such patients at an earlier date is that rates of decline of FEV1 and DLCO are variable The aims of our study were to determine whether measurement of four or more FEV1 or DLCO performed over 18 months could predict future declines in lung function, which might prompt earlier initiation of mTOR inhibitor therapy. The rationale to evaluate in addition a treatment criteria based on rates of decline of DLCO and a DLCO 70% pred was prompted by those patients who have FEV1 >70% pred, and experience persistent decline in DLCO. Inasmuch as such patients were not the subject of the MILES trial, we thought that excluding LAM patients with reduced DLCO from this analysis appeared to be inappropriate. The value of rates of change in forced expiratory volume in 1 s (FEV1) and diffusing capacity of the lung for carbon monoxide (DLCO) to predict disease progression, and initiation of mTOR (mechanistic target of rapamycin) inhibitor therapy has not been evaluated. In 84 premenopausal LAM patients, individual rates of change in FEV1 and DLCO and their 95% confidence intervals were used to derive subsequent lowest values of FEV1 and DLCO that would prompt initiation of sirolimus therapy. These treatment criteria were compared with a criterion based on FEV1 or DLCO 70% predicted. In 12 patients undergoing sirolimus therapy both methods for determining the optimal point for initiation of therapy were evaluated. 27 and 35 patients who experienced greater than expected rates of change in FEV1 and DLCO, respectively, would have been excluded from therapy based on an FEV1 or DLCO >70% pred. Twenty five of the 84 patients were eventually treated, but only when FEV1 or DLCO were 70% pred. Applying such treatment criteria to 12 patients undergoing sirolimus therapy would have delayed treatment for many years. Premenopausal females in whom FEV1 or DLCO are declining at rates above the expected based on their individual rates of decline, should be considered for sirolimus therapy before the FEV1 or DLCO falls to 70% pred. 2. Pseudoneutropenia in lymphangioleiomyomatosis (LAM) patients receiving sirolimus: evaluation in a 100 patient cohort Diurnal variation in white blood cells (WBC), particularly neutrophils, is well-described. WBC levels are lower in the morning and increase through the day. Drugs with immunosuppressive effects, such as sirolimus, may further lower WBC counts. This phenomenon has been observed in clozapine and related atypical antipsychotic medications, drugs with known immunosuppressive effects. For patients receiving these drugs, blood counts measured in the early morning may lead to a false impression of low WBC/neutrophil counts (pseudoleukopenia/pseudoneutropenia) that may result in discontinuation or a reduction in dose and suboptimal treatment. Of importance, isolated morning neutropenia is not known to increase the risk of infection. LAM is treated with inhibitors of mechanistic target of rapamycin (mTOR), such as sirolimus. Sirolimus is an immunosuppressive agent that has been approved by the Food and Drug Administration for use in transplant recipients and has been recently approved for use in LAM. In a study evaluating the sustained effects of sirolimus in LAM, neutropenia/leukopenia was reported in 40% of patients. Diurnal variation may not be appreciated in the interpretation of low WBC counts. We report here a patient with LAM receiving sirolimus who presented in the morning with low WBC and neutrophil counts. Previously, neutropenia caused the primary physician to decrease the dose of sirolimus. Repeat of the cell counts later in the day showed an increase in WBC and neutrophil counts by 42%. These WBC and neutrophil levels would not warrant modification of the sirolimus dose. The objective of this study was to determine if LAM patients experience pseudoleukopenia and/or pseudoneutropenia, particularly when they are on sirolimus treatment, and whether this phenomenon is associated with increased incidence or severity of infection. We examined a cohort of 100 LAM patients either treated or not treated with sirolimus. We compared leukocyte counts at three time-points throughout the day. Since diurnal effects are also affected by food intake, we measured leukocyte counts prior to meals and following breakfast and lunch. In patients that experienced pseudoleukopenia and/or pseudoneutropenia, incidence and severity of infection were collected up to 1 year prior to the study date and up to 1 year after the study date. 100 female patients with LAM were provided a breakfast and lunch after a fasting period of 7 h. Blood samples were taken at three times: morning (06:00 h 1.5 h) prior to breakfast, mid-day (11:00 h 1.5 h) about 2 h after breakfast, and afternoon (15:00 h 1.5 h) about 1 h after lunch. A CBC with differential and lipid panel was obtained at each measurement. ANOVA was used to analyze results within and between patients and, in addition, based on sirolimus use (55 patients receiving sirolimus, 45 patients not receiving sirolimus). Among patients receiving sirolimus, 16.4% (nine out of 55 patients) presented with pseudoleukopenia, while 2.2% of patients not receiving sirolimus (one out of 45 patients) presented with pseudoleukopenia. 7.3% of patients receiving sirolimus (four out of 55 patients) presented with pseudoneutropenia, while 8.89% of patients not recieiving sirolimus (four out of 45 patients) presented with pseudoneutropenia. However, neutrophil counts were 35% lower in the morning in patients on sirolimus experiencing pseudoneutropenia compared to patients off treatment experiencing pseudoneutropenia. Treatment status significantly affected variation in WBC and neutrophil count between the morning and mid-day/afternoon. Among the 14 patients who experienced pseudoneutropenia/pseudoleukopenia, the severity of all reported infections was mild. Rates of infection between patients receiving sirolimus and not treated with sirolimus were similar. In the larger patient cohort (n=100 patients), absolute counts for WBC, neutrophils and monocytes were lowest in the morning, and increased later in the day. Eosinophil counts decreased from the morning to the early afternoon. Significant changes in monocyte count throughout the day were only observed in patients on sirolimus. Lymphocyte, basophil and immature granulocyte counts did not show significant variation. Thus, some LAM patients on sirolimus experience pseudoleukopenia and pseudoneutropenia. In patients experiencing pseudoleukopenia or pseudoneutropenia, infection rates were low and similar.